IDENTIFICATION OF TYROSINE KINASE AND SERINE/THREONINE KINASE ACTIVITIES IN STAPHYLOCOCCUS AUREUS ATCC12600

摘要

Protein phosphorylation is one of the key mechanisms which regulate colonization, pathogenesis, antibiotic resistance and various metabolic pathways in Staphylococcus aureus. Therefore, the present study is focused on the molecular characterization of Serine-Threonine Phospho Kinase (STPK) and bacterial tyrosine kinase (BYK) in Staphylococcus aureus ATCC12600. In this context we have developed a unique phosphorylation methodology to identify the STPK and BYK. These enzymes have both substrate level phosphorylation and autophosphorylation properties; they basically phosphorylate substrates having S and T residues and Y residues using gamma phosphate of ATP as the phosphate donor. These phosphorylated enzymes and synthetic peptides (HGLDNYRGYSLG substrate for BYK NLCNIPCSALLSSDITASVNCAK substrate for STPK) were fractionated on Sephadex G-25 column and the pure phosphorylated proteins obtained were treated with Reagent and the bound phosphate was measured at 820nm. These phosphorylated proteins were fractionated in SDS-PAGE and detected by immersing the gel in reagent.